Breast cancer comprises a heterogeneous group of diseases that vary in morphology, biology, behavior and response to therapy. Among women, breast cancer remains the second most important cause of death (by cancer). Patients who cannot be cured are those in whom breast cancer has metastasized, that is, breast cancer cells have migrated and invaded other organs such as lung and bone.
Triple-negative breast cancer (estrogen-receptor-α (ER)-negative, progesterone-receptor (PR)-negative, and human epidermal growth factor 2 receptor (HER2)-non overexpressed) is a subtype of breast cancer that accounts for approximately 15% of breast cancer. Triple-negative breast cancer (TNBC) is a subtype of tumor known for its aggressive clinical behavior.
Triple negative breast cancer and endocrine-resistant breast cancer tumors are an important area of research for both researchers and clinicians alike for being poor prognostic factors for disease-free and overall survival. Besides, no effective specific targeted therapy is readily available therefor.
Previous studies have identified an acyl-CoA synthetase 4 (ACSL4) gene-expression pattern correlated with triple-negative tumors. It has been shown that both in breast cancer cell lines and in tumor samples the expression of acyl-CoA synthetase 4 (ACSL4) is inversely correlated with ER levels. ACSL4 belongs to a five-member family of enzymes that esterifies mainly arachidonic acid (AA) into acyl-CoA (Maloberti P. M. et al., 2010; Functional interaction between acyl-CoA synthetase 4, lipooxygenases and cyclooxygenase-2 in the aggressive phenotype of breast cancer cells. PLoS One 5, e15540; Orlando, U. D. et al., 2012; The functional interaction between Acyl-CoA synthetase 4, 5-lipooxygenase and cyclooxygenase-2 controls tumor growth: a novel therapeutic target. PLoS One 7, e40794.) and, unlike the other ACSL isoforms, ACSL4 is encoded on the X chromosome and its expression is highest in adrenal cortex, ovary and testis (Kang, M. J. et al., 1997; A novel arachidonate-preferring acyl-CoA synthetase is present in steroidogenic cells of the rat adrenal, ovary, and testis. Proc Natl Acad Sci USA 94, 2880-2884.). ACSL4 is also highly expressed in mouse and human cerebellum and hippocampus. The physiological functions of ACSL4 have been studied and include possible roles in polyunsaturated fatty acid metabolism in brain, in steroidogenesis and in eicosanoid metabolism related to apoptosis (Maloberti P. M. et al., 2005; Silencing the expression of mitochondrial acyl-CoA thioesterase I and acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis. Febs J 272, 1804-1814.). ACSL4 expression has also been associated with non-physiological functions such as mental retardation disorder (Modi, H. R. et al., 2013; Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid, uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: a potential drug for bipolar disorder. Biochim Biophys Acta 1831, 880-886.) and cancer (Maloberti P. M. et al., 2010, supra). ACSL4 was first associated with cancer due to its abnormal expression in colon and hepatocellular carcinoma. Increased ACSL4 expression, both at mRNA and protein levels, in colon adenocarcinoma cells has been associated with the inhibition of apoptosis and an increase in cell proliferation when compared to adjacent normal tissue. ACSL4 has also been suggested as a predictive factor for drug resistance in breast cancer patients receiving adriamycin-containing chemotherapy.
The present inventors have demonstrated a positive correlation of ACSL4 expression and aggressiveness in breast cancer cell lines, with the highest expression found in metastatic lines derived from triple-negative tumor breast cancer (MDA-MB-231 and Hs578T) (Maloberti P. M. et al., 2010, supra). Functionally, it was found that ACSL4 is part of the mechanism responsible for increased breast cancer cell proliferation, invasion and migration, both in vitro and in vivo (Maloberti P. M. et al., supra, 2010; Orlando U. D. et al., 2012, supra). Accordingly, the sole transfection of MCF-7 cells, a model of non-aggressive breast cancer cells, with ACSL4 cDNA transforms them into a highly aggressive phenotype, and it was further demonstrated that ACSL4 can be silenced to reduce cell line aggressiveness. Furthermore, the stable transfection of MCF-7 cells with ACSL4 using the tetracycline Tet-Off system (MCF-7 Tet-Off/ACSL4) and their injection into nude mice has resulted in the development of growing tumors with marked nuclear polymorphism, a high mitotic index and low expression of ER and PR (Orlando U. D. et al., 2012 supra), all of which demonstrates the transformational capacity of ACSL4 overexpression. The role of ACSL4 in the development of growing tumors found further support when tumor growth was inhibited through the inhibition of ACSL4 expression by treating mice with doxycycline. Although the role of ACSL4 in mediating the aggressive phenotype in breast cancer is well accepted, the mechanism involved in this effect has yet to be fully elucidated. And, as enzyme overexpression can solely change cell phenotype from mildly aggressive to highly aggressive, the MCF-7 Tet-Off/ACSL4 model may be regarded as a valuable technique to study the mechanisms through which ACSL4 triggers the phenotype change.
The idea of personalized medicine and molecular profiling for prognostic tests has led to a plethora of studies in the past 10 years, in search for genetic determinants of metastatic breast cancer. Such studies have identified gene sets, or “signatures”, whose expression in primary tumors is associated with higher risk of metastasis and poor disease outcome for the patients.
The present application discloses experimental evidence of the role played by the ACSL4 overexpression in the aggressive phenotype of TNBC.
Although the role of ACSL4 in mediating an aggressive phenotype in breast cancer is well accepted, there is little evidence as to the early steps through which ACSL4 increases tumor growth and progression. Therefore, the present inventors performed a massive in-depth mRNA sequencing approach and the reverse-phase protein array using MCF-7 Tet-Off/ACSL4 as a model to identify gene expression and functional proteomic signatures specific to ACSL4 overexpression. In particular, the present inventors make use of the tetracycline Tet-Off system to stably transfect non-aggressive breast cancer MCF-7 cells and develop a stable line overexpressing ACSL4 (MCF-7 Tet-Off/ACSL4). As a result, the present inventors have proven that cell transfection solely with ACSL4 cDNA renders a highly aggressive phenotype in vitro and results in lower ER expression and the development of growing tumors when injected into nude mice.
The sole expression of ACSL4 displays a distinctive transcriptome and functional proteomic profile, and results show that the most significantly up-regulated gene networks in breast cancer cells overexpressing ACSL4 include genes associated with the regulation of embryonic and tissue development, cellular movement and DNA replication and repair.
In addition, the present inventors have shown in previous studies that the effects of Rosiglitazone on cell and tumor growth in vitro or in vivo are similar to those obtained with the specific inhibition of ACSL4 by doxycycline treatment of the MCF-7 Tet-Off/ACSL4 (Orlando U. D. et al., 2012, supra), the minimal doses exerting significant inhibitory effects being 75 μM for rosiglitazone.
Rosiglitazone, a member of the thiazolidinedione family of drugs (TZDs), is known to attenuate cell growth in carcinoma of various organs including breast, prostate, lung, colon, stomach, bladder and pancreas. Rosiglitazone and derivatives of troglitazone have been used either alone or in combination in experimental conditions to inhibit the growth of different tumor cell lines (Luconi, M. et al., 2010; Rosiglitazone impairs proliferation of human adrenocortical cancer: preclinical study in a xenograft mouse model. Endocr Relat Cancer 17, 169-177.) and, although the action of rosiglitazone has been attributed to its effects on the peroxisome proliferator-activated receptor gamma, in vitro studies performed with rat recombinant proteins have demonstrated that TZDs can directly inhibit the activity of one of the gene products of the acyl-CoA synthetases, i.e. ACSL4.
Rosiglitazone is an antidiabetic drug in the thiazolidinedione class of drugs. It works as an insulin sensitizer, by binding to the PPAR receptors in fat cells and making the cells more responsive to insulin. Despite rosiglitazone's effectiveness at decreasing blood sugar in type 2 diabetes mellitus, at daily oral dose in the range of 4 to 8 mg, its use decreased dramatically as studies showed apparent associations with increased risks of heart attacks and death. On Sep. 23, 2010 the US Food and Drug Administration issued a decision to restrict access to rosiglitazone medicines. In Europe, the European Medicines Agency (EMA) recommended in September 2010 that the drug be suspended from the European market because the benefits of rosiglitazone no longer outweighed the risks.
Another member of the drug class of thiazolidinediones, Troglitazone, a peroxisome proliferator-activated receptor gamma agonist, which enhances insulin sensitivity, was approved for the treatment of type 2 diabetes in 1997. Troglitazone was available in 400 mg tablets. The recommended dosage was 400 to 800 mg once daily. However, within a year after its widespread use, individual cases of liver injury and failure were reported, leading to the withdrawal of troglitazone from the market in the year 2000.
According to the findings of the present inventors, an ACSL4 overexpression gene and functional proteomic signature was derived which might reveal important information about novel mediators of breast cancer cell aggressiveness. By means of a model of ACSL4 overexpression and a pharmacological approach, it was also showed that ACSL4 and the mTOR pathway from the transcriptome and functional proteomic profile are functionally required and work in a synergistic way for cell proliferation in the MCF-7 Tet-Off/ACSL4 model.
It is also demonstrated herein that ER expression is down-regulated and that specific pathways such as AKT-mTOR-SP6 kinase and Wnt (Wingless-Type MMTV Integration Site Family) are functionally required for ACSL4 action.
Rapamycin, also known as sirolimus, is an mTOR inhibitor macrolide, originally identified in sirolimus-resistant mutants of Saccharomyces (Vézina C. et al., 1975; Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle. J Antibiot (Tokyo). 1975 October; 28(10):721-6.). Though it was first developed as an antifungal agent, its immunosuppressive and antiproliferative properties later redirected its use towards the treatment of certain tumors.
Rapamycin and its analogues are being used in clinical trials as novel-targeted anticancer agents and although their activity in this context has been proven, results show that only some of the treated patients actually respond to treatment (Noh W. C. et al., 2004; Determinants of rapamycin sensitivity in breast cancer cells. Clin Cancer Res 10, 1013-1023).
In conclusion, ACSL4 is an upstream regulator of tumorigenic pathways and the data herein provide novel insights into a combined pharmacological approach. Because an aggressive tumor phenotype appears in the early stages of metastatic progression, the previously unknown mediators of ACSL4 might become valuable prognostic tools or therapeutic targets in breast cancer.